Study On Cyanophage Diversity And Isolation And Identification Of Eutrophic Lakes In Yunnan Province

Cyanophage specifically infects cyanobacteria and is widely found in freshwater and marine environments.Cyanophage can infect cyanobacteria in different time and space,change the host population structure,regulate the abundance,diversity and community structure succession of cyanobacteria.Cyanophage is an important part of the aquatic microbial community and is rich in genetic diversity.It has great potential to control the massive reproduction caused by cyanobacteria.Cyanophage diversity and its ecological role have received much attention.Studying the diversity of cyanophages helps to understand the relationship between cyanophages and host cyanobacteria,thereby promoting the control of cyanobacteria blooms.In this study,different eutrophic freshwater lakes in Yunnan plateau(Dianchi Lake,Xingyun Lake and Fuxian Lake)were selected as the research objects.Based on different marker genes of cyanophage,the diversity of different groups of cyanophage genes in water and sediment of three lakes in the autumn and winter of 2018 was studied.The genetic diversity of different phage genes in each lake was analyzed.The biological characteristics,morphology and molecular biology of infected cyanophages were analyzed.The results of the study are helpful to analyze the evolution process of cyanophage,improve its classification,and further explore the evolutionary relationship between cyanophages,and provide reference for further understanding of the micro-ecological system of eutrophic lakes in Yunnan.The main contents and results of the study are as follows1.Different molecular marker genes of cyanophage were amplified in lake water and sediment.PsbA gene and g20 gene were amplified successfully in Dianchi Lake.g20 gene and MazG gene were amplified successfully in Xingyun Lake water samples.Four genes in Fuxian Lake water samples were amplified successfully,namely psbA gene,g20 gene,MazG gene and phoH gene.Only the psbA gene was successfully amplified in sediments of three lakes,but no other genes were successfully amplified.2.The genetic diversity of cyanophage in eutrophic lakes Dianchi Lake,Xingyun Lake and Fuxian Lake was analyzed based on the psbA gene.In autumn(September-November)and winter(December-February)of 2018,86 psbA sequences of cyanophage were found in water samples of Dianchi Lake,Xingyun Lake and Fuxian Lake,including 25 in Dianchi Lake,27 in Xingyun Lake and 34 in Fuxian Lake.Cyanophage psbA sequences obtained from three lakes and reference sequences were used to construct phylogenetic tree.The phylogenetic tree showed that the phylogenetic psbA sequences obtained in this study were more similar to those obtained from freshwater.The phylogenetic psbA gene diversity was rich in Fuxian Lake and Dianchi Lake in autumn and winter of 2018,and the sequence of water samples in autumn and winter was different.The sequence of Xingyun Lake was different from that of Dianchi Lake and Fuxian Lake.Sequences are quite different and diversity is not abundant.3.The genetic diversity of cyanophage in eutrophic lakes Dianchi Lake,Xingyun Lake and Fuxian Lake was analyzed based on the cyanophage g20 gene.A total of 82 effective cyanophage g20 gene sequences were obtained,including 31 in Dianchi Lake,21 in Xingyun Lake and 30 in Fuxian Lake.A phylogenetic tree was constructed by using g20 sequences and reference sequences obtained from three lakes.The results showed that the g20 gene of Fuxian Lake was rich in diversity,and most of the sequences had high homology with the uncultured phage g20 sequence of French freshwater lake and East Lake of China,but the difference was not significant in autumn and winter.Dianchi g20 gene is also rich in diversity,most of which is in the middle.The uncultured phage g20 sequences of rice in Northeast China and Lake Bourges in France are highly homologous,with some differences in autumn and winter sequences.However,the genetic diversity of Xingyun Lake is lower than that of the other two lakes,and it has high homology with the uncultivated algal g20 sequence in the Pacific environment,and the sequence in autumn and winter is not much different.4.A cyanophage was isolated from the concentrated water samples of Dianchi Lake through double-layer plate purification experiments,which could stably infect the Phormidium and expand its cultivation under laboratory conditions.The cyanophage curve of the infected host cyanobacteria first increased and then decreased.The phage-dissolving cyanobacteria is concentrated for 4-7 days.The diameter of the phagocytic body was about 50 nm,and the tail was observed by TEM.The psbA gene was successfully amplified from purified phage DNA and compared by sequencing with the psbA gene as a source of phages.5.The biological characteristics of cyanobacteria indicate that phage stored at 4 °C has strong infectious activity without any protective agent,and the infectious activity can last for one year.The phage stored at-80 ° C completely lost its infectious activity.After acting at 40 ° C,the cyanobacteria have a certain infectious activity,and the phage treated at 50 ° C or above loses its infectious activity.Within 20 minutes after UV irradiation,the infection activity decreased with the prolongation of the irradiation time;after ultraviolet irradiation for more than 30 minutes,the infection activity was lost;the algae liquid was cleared after the infection of cyanophage treated with CHCl3,and the control groups of terile water was cleared after the treatment of CHCl3.It was inferred that CHCl3 affected the growth of algae cells and even caused the death of algae cells.